MX2007013719A

MX2007013719A - Abfb-1 gene of penicillium funiculosum.

Info

Publication number: MX2007013719A
Application number: MX2007013719A
Authority: MX
Inventors: Olivier Nore; Jean Marie Francois; Jean-Luc Parrou; Olivier Tourrasse
Original assignee: Adisseo France Sas
Priority date: 2005-05-04
Filing date: 2006-05-03
Publication date: 2008-01-24
Also published as: FR2885366A1; AU2006243138C1; ZA200709272B; JP2008539708A; MY142808A; PL1877561T3; CA2607757C; AU2006243138A1; KR20080016829A; AR053376A1; JP5048651B2; AU2006243138B2; FR2885366B1; EP1877561A1; SI1877561T1; CN101171336A; CA2607757A1; KR101276752B1; DK1877561T3; TWI368654B

Abstract

The invention relates to the abfB-1 gene of Penicillium funiculosum that codes for a type B ????-L-arabinofuranosidase. This enzyme ????-L-arabinofuranosidase can be incorporated in nutritional additives or in foods for animals for which it improves the digestibility and thus the nutritional value.

Description

FIELD OF THE INVENTION The invention relates to the isolated abfB-1 gene of Penicillium funiculosum and to the ABFB-1 polypeptide encoded by this gene having an activity -L-arabinofuranosidase B. BACKGROUND OF THE INVENTION The Penicillium funiculosum is a Talaromyces that belongs to the Aspergilleae family. The isolation of this microorganism from numerous organic substrates that are subject to air or water contamination shows that this fungus has a range of hydrolytic enzymes of surprising richness. The use of this enzymatic cocktail in animal feed contributes to the depolymerization of natural organic substances and makes it possible to improve their digestibility. WO 99/57325 thus describes a strain of Penicillium funiculosum called IMI378536 that produces a mixture of enzymes that is particularly suitable as animal feed. However, the enzymatic cocktails produced by Penicillium funiculosum have not been characterized to a large extent biochemically. In fact, only a limited number of enzymatic activities, such as xylanases and β-glucanases are generally measured in the obtained fermentation broths. These activities reflect only a fraction of the enzymatic population present in Ref .: 187036 the cocktail. The hemicellulolytic compounds derived from agriculture constitute the second reserve of polysaccharides after cellulose in vegetable tissues. This group is characterized by a wide variety of heteropolysaccharides, of which the main representatives are xylans, arabinans, galactans, glucans and mannans. The arabinose in its furfural form is widely represented among heteropolysaccharides such as arabinanos and arabinoxylans. Arabinano is a polymer with arabinofuranose residues bound by a-1-5 bonds and can be substituted with 1 or 2 arabinose residues in the 0-2 or 0-3 position. With respect to arabinoxylans, a-L-arabinofuranosyl residues are linked to the β-4-xylopyranosyl backbone by a-1-3 and a-1-2 bonds. The presence of arabinose residues in these side chains can restrict the enzymatic hydrolysis of the hemicellulolytic compounds in numerous industrial applications such as the increased digestibility of animal feed. The enzymes that divide the a-L-arabinofuranoside bonds can act in synergy with the xylanases to allow hydrolysis of arabinoxylans and arabinanos. The arabinase activities (endo-, exo-arabinase and, predominantly, a-L-arabino-furanosidase activities) can therefore contribute, actively and synergistically, with the xylanases, to the depolymerization of hemicellulolytic compounds. Hemicellulolytic and pectic compounds can represent up to 50% of the total carbohydrates present in plants and are a major source of energy for animals. The increase in, the digestibility of these compounds is correlated with the decrease in the degree of substitution of arabinosyl residues within the hemicellulolytic compounds (Brice, RE, Morrison, IM, 1982, Carbohydr.Res. 101: 93-100) . Enzymes that hydrolyze the bonds between L-arabinose residues have been isolated from microorganisms such as bacteria or filamentous fungi. The arabinosidases consist mainly of a-L-arabinofuranosidases (EC 3.2.1.55) which are capable of hydrolyzing non-reducing L-arabinofuranosyl residues derived from L-arabinoxylan or compounds such as arabinanos and arabinogalactans. The α-L-arabinofuranosidases (EC 3.2.1.55) have been classified into two families of glycoside hydrolases (GH 51 and GH 54) according to their protein sequence similarities. These two families differ by virtue of their specificity for the substrate contained in the polysaccharides. The first group (GH 51) contains type A arabinofuranosidases that act only on small linear structures of linked arabinofuranosyl oligosaccharides -1-5. The second group consists of type B arabinofuranosidases (GH 54) that catalyze the hydrolysis of the a-1 bonds, 5, a-1,3 and a-1,2 of the side chains contained in the arabinofuranosyl-oligosaccharide compounds. B arabinofuranosidases (ABFB) have been isolated from numerous bacteria, but also from filamentous fungi. The genus Aspergillus is the one that is most represented, but they have also been isolated from the genera Trichoderma, Penicillium and Fusarium. WO 96/29416, WO 96/06935, WO 2004/018662 and US 5,989,887 describe arabinofuranosidase genes from Aspergillus niger. The alignment of protein sequences shows that the abfB protein of A. niger is 72.4% identical to the protein ABFB-1 of P. funiculosum. No essential characteristics are given with respect to the use of the polypeptide in animal feed in these patent applications. Clinche et al. (J. Agrie, Food Chem., 45, 2379-2383, 1997) have described three a-L-arabinofuranosidases from Aspergillus terreus that have a potential application in oenology. Gielkens et al. (Microbiology, 145, 735-741, 1999) the abfb gene of Aspergillus nidulans has been described. The abfB genes of Aspergillus ka achii and Aspergillus awamori have been described by Koseki et al. (J. of Bioscience and Bioengineering, Vol. 96, No. 3, 232-241, 2003). These enzymes have applications in the fermentation of the Japanese Shochu liqueur. The abfB gene of the filamentous fungus Trichoderma reesei has been described by Margolles-Clark et al. (Applied and Environmental Microbiology, 3840-3846, 1996). Panagiotou et al. they also described two extracellular alpha-L-arabinofuranosidases derived from Fusarium oxysporum (Can. J. Microbiol., 2003: 49 (10): 639-4). Carvallo et al. (Mycol. Res., 107 (4), 388-394, 2003) have described B a-L-arabinofuranosidase from Penicillium purpurogenum. The alignment of protein sequences shows that the abf-1 protein of P. purpurogenum is 85.6% identical to the ABFB-1 protein of P. funiculosum. In this article, essential characteristics are not given regarding the use of the polypeptide in animal feed. Sakamoto et al. (FEBS Letters 560, 199-204, 2004) have described the abnx gene of Penicillium chrysogenum which, however, codes for an arabinase activity other than ABFB activity. However, these ABFB enzymes do not have the optimum qualities required for application in animal feed. Actually, to be able to use them in animal feed, the ABFBs have to have properties compatible with the treatments to which the animal feed intended for this food is subjected. In particular, the activity of the enzymes used must be stable under the processing temperature and the pH conditions, and, if possible, be optimal in the preparation of these animal feeds and under the conditions that exist in the digestive system of animals that ingest these animal feeds. In addition, these enzymes have to have a broad spectrum of action (debranching) on the arabinano heteropolysaccharides, arabinoxylans and arabinogalactans) to allow an effective increase in the digestibility of animal feed by animals. This increase in the digestibility of animal feed makes it possible to increase its nutritional value. Accordingly, enzymes having increased specificity (stereospecificity, enantioselectivity), activity or affinity with respect to the natural substrates arabinoxylans and arabinans are of great interest as animal feeds. The present invention describes an L-arabinofuranosidase B (ABFB-1) of Penicillium funiculosum suitable for application in animal nutrition and the gene encoding this enzyme. The invention also relates to the homologs, variants and fragments of ABFB-1 which retain the same - 1 - catalytic properties. Advantageously, the ABFB enzymes according to the invention have a high optimum temperature. Another advantage of the present invention is that the expression of ABFB-1 from Penicillium funiculosum is induced to a high degree naturally in this fungus under conditions for the induction of cellulolytic and hemicellulolytic enzymes (industrial type culture medium for the production of cellulolytic enzymes and hemicellulolytics). The enzymes according to the invention also have other industrial or agroindustrial applications. In particular, the treatment of fruit juices can be mentioned, the manufacture of paper, the conversion of hemicellulolytic biomass into fuel or chemical products, the preparation of alcoholic beverages by fermentation. Sequence description SEQ ID No. 1: Genomic sequence of the abfB-1 gene of Penicillium funiculosum. SEQ ID No. 2: Sequence of Penicillium funiculosum ABFB-1 polypeptide having a-L-arabinofuranosidase type B activity. SEQ ID No. 3: Xbal-abfB primer. SEQ ID No. 4: HindIII-abfB primer. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a polypeptide adapted for use in animal feed comprising a polypeptide chosen from the following polypeptides: the polypeptide of SEQ ID No. 2, the polypeptide whose sequence is between the position 28 and position 507 of SEQ ID No. 2, a fragment of the polypeptide of SEQ ID No. 2 having an α-arabinofuranosidase B activity, a polypeptide having an α-arabinofuranosidase B activity and having an identity of at least 90% with the polypeptide of SEQ ID No. 2. The invention also relates to a polynucleotide, which encodes an aL-arabinofuranosidase B activity, chosen from the following polynucleotides: the polynucleotide whose sequence is between position 845 and position 2368 of SEQ ID No. 1, the polynucleotide whose sequence is between position 927 and position 2368 of SEQ ID No. 1, a polynucleotide leotide encoding a polypeptide according to claim 1. Another object of the present invention is a polynucleotide having the sequence represented by SEQ ID No. 1 or the sequence complementary to SEQ ID No. 1. The invention also relates to to expression cassettes comprising, in the direction of transcription: - a promoter that is functional in a host organism; a polynucleotide according to the invention; and a terminator sequence that is functional in the same host organism. Another object of the invention is a vector comprising a polynucleotide according to the invention and / or an expression cassette according to the invention. The invention also relates to a host organism transformed with a polynucleotide according to the invention, to an expression cassette according to the invention and / or to a vector according to the invention. In one embodiment of the invention, the host organism is chosen from yeasts and filamentous fungi. Preferably, the host organism is a strain of Penicillium funiculosum. The invention also relates to a nutritional additive for animals, comprising a polypeptide according to the invention, a host organism according to the invention or a fermentation broth of a host organism according to the invention. Preferably, this nutritional additive is in liquid form or in powder form. Another aspect of the invention is an animal feed comprising a nutritional base for animals and a nutritional additive for animals according to the invention. The invention also relates to the use of an ABFB polypeptide according to the invention or to a host organism according to the invention for the preparation of a nutritional additive for animals or an animal feed. Another object of the invention is the use of a polypeptide ABFB according to the invention or a host organism according to the invention for hydrolyzing the α-L-arabinofuranosyl bonds of the arabinofuranosyl-oligosaccharide compounds. Polypeptides The present invention therefore relates to ABFB polypeptides having an α-L-arabinofuranosidase B activity. Preferably, these polypeptides are isolated from Penicillium funiculosum. The expression "aL-arabinofuranosidase B" means aL-arabinofuranosidases (EC 3.2.1.55) type B (GH 54) that catalyze the hydrolysis of a-1,5, a-1,3, and a-1,2 bonds of the side chains contained in the arabinofuranosyl-oligosaccharide compounds. The α-L-arabinofuranosidase B of strain 1M1378536 of Penicillium funiculosum is represented in SEQ ID No. 2.

The term "polypeptide adapted for use in animal feed" means a polypeptide whose characteristics are such that it is suitable for feeding animals. The essential characteristics for feeding animals are particularly the pH and the temperature at which the enzyme is active. In fact, the digestive system of animals is acidic and therefore it is essential that the enzyme remains active at this type of pH, to maintain its activity in the hydrolysis of L-arabinose residues. In addition, the formulation of the enzyme in a nutritional additive or in animal feeds comprises treatments and special conditions, particularly a temperature above room temperature. The activity of the enzyme must therefore be stable under these formulation conditions, particularly the temperature conditions. In one embodiment of the present invention, the polypeptide exhibits an α-arabinofuranosidase B activity at an acid pH, for example, at a pH greater than 5, preferably at a pH greater than 4. According to a preferred embodiment of the present invention , the polypeptide has an optimal α-arabinofuranosidase B activity between pH 2 and pH 3.5. In a preferred embodiment of the present invention, the polypeptide exhibits an α-L-arabinofuranosidase B activity at a temperature above room temperature. Preferably, the polypeptide of the present invention exhibits an optimal α-L-arabinofuranosidase B activity at a temperature comprised between 40 and 70 ° C, more preferably between 50 and 65 ° C. In a preferred embodiment, the polypeptides according to the invention are glycosylated. The polypeptide of SEQ ID No. 2 possesses in particular N-glycosylation sites at amino acid 92 and amino acid 376. In a preferred embodiment, the asparagine residues at position 92 and at position 376 of the polypeptide of the SEQ ID No. 2 are glycosylated. The a-L-arabinofuranosidase B of Penicillium funiculosum is an enzyme secreted by the fungus in its extracellular environment. The polypeptide of SEQ ID No. 2 therefore comprises a signal peptide of 27 amino acids. The object of the invention is also the mature polypeptide obtained after dividing the signal peptide. In particular, the invention relates to the polypeptide whose sequence is between position 28 and position 507 of SEQ ID No. 2. In another embodiment, the signal peptide of the polypeptide of SEQ ID No. 2 can be replaced by a heterologous signal peptide for the expression and secretion of the polypeptide of SEQ ID No. 2 by a heterologous host organism. The invention also relates to fragments of the polypeptide of SEQ ID No. 2 having an α-L-arabinofuranosidase B activity. The term "fragment" of a polypeptide denotes a polypeptide comprising a part but not all of the polypeptide of which is derived. The invention therefore relates to a polypeptide comprising a fragment of at least 100, 200, 300, 400 or 500 amino acids of the polypeptide of SEQ ID No. 2. This fragment of the polypeptide of SEQ ID No. 2 preserves its α-arabinofuranosidase activity B. The invention it therefore refers to the biologically active fragments of the polypeptide of SEQ ID No. 2. The term "biologically active fragment" denotes a fragment of a polypeptide which retains the function of the polypeptide from which it is derived. The biologically active fragments of the polypeptide of SEQ ID No. 2 thus preserve the function of the ABFB-1 polypeptide of Penicillium funiculosum. These biologically active fragments have an α-L-arabinofuranosidase B activity. Methods for preparing fragments of a polypeptide and techniques for measuring α-L-arabinofuranosidase B activity are well known to one skilled in the art. The subject of the invention are also polypeptides having an L-arabinofuranosidase B activity and having at least 90% identity with the polypeptide of SEQ ID No. 2. Preferably, these polypeptides have the same properties and in particular the same catalytic properties as the polypeptides of SEQ ID No. 2.

Preferably, these polypeptides are isolated from other strains of Penicillium funiculosum or other filamentous fungi. Alternatively, these polypeptides can be obtained, for example, by site-directed mutagenesis techniques. The object of the invention is a polypeptide having at least 90%, 95%, 98% and preferably at least 99% of amino acids that are identical to the polypeptide of SEQ ID No. 2. The expression "identical amino acids" means amino acids that they do not vary or do not change between two sequences. These polypeptides may have a deletion, addition or substitution of at least one amino acid compared to the polypeptide of SEQ ID No. 2. The object of the invention is also a polypeptide having at least 90%, 95%, 98% and preferably at least 99% similarity to the polypeptide of SEQ ID No. 2.

The similarity of the expression means the measurement of the similarity between the protein or nucleic sequences. These polypeptides can have a deletion, an addition or a substitution of at least one amino acid compared to the polypeptide of SEQ ID No. 2. The degree of similarity between two sequences, quantified by a result, is based on the percentage of identity of the sequences and / or substitutions that preserve the sequence.

Methods for measuring and identifying the degree of identity and degree of similarity between polypeptides are known to those skilled in the art. It is possible to use, for example, the vector NTi 9.1.0, the alignment program AlignX (Clustal W algorithm) (Invitrogen INFORMAX, http: //. Invitrogen. Com). Preferably, the default parameters are used. The polypeptides according to the invention are isolated or purified from their natural environment. The polypeptides can be prepared by various methods. These methods are, in particular, purification of natural sources such as cells that naturally express these polypeptides, the production of recombinant polypeptides by appropriate host cells and their subsequent purification, production by chemical synthesis or, finally, a combination of these various proposals. These various production methods are well known to those skilled in the art. Thus, the ABFB polypeptides of the present invention can be isolated from Penicillium funiculosum. In another embodiment, the ABFB polypeptides of the present invention are isolated from recombinant host organisms that express an ABFB polypeptide according to the invention. Object of the invention are also fusion proteins, recombinant proteins or chimeric proteins comprising the polypeptides according to the invention. The term "polypeptide" also denotes modified proteins and polypeptides. The polypeptides according to the invention have an ABFB activity and preferably retain the catalytic properties of the enzyme ABFB-1 of Penicillium funiculosum.

In particular, these polypeptides have an optimal activity at 60 ° C and at pH 3.4. Polynucleotides The invention also relates to polynucleotides that encode an α-arabinofuranosidase B activity. According to the present invention, the term "polynucleotide" means a single-stranded nucleotide chain or its complementary strand which may be of the DNA or RNA type, or a chain of double-stranded nucleotides that can be of the genomic or complementary DNA type. Preferably, the polynucleotides of the invention are of the DNA type, in particular double-stranded DNA. The term "polynucleotide" also denotes the modified polynucleotides. The polynucleotides of the present invention are isolated or purified from their natural environment. Preferably, the polynucleotides of the present invention can be prepared by conventional molecular biology techniques as described by Sambrook et al. ("Molecular Cloning: A Laboratory Manual", 1989) or by chemical synthesis.

In a first embodiment, the invention relates to the polynucleotide whose sequence is between the position 845 and position 2368 of SEQ ID No. 1. This polynucleotide encodes the enzyme ABFB-1 of Penicillium funiculosum of SEQ ID No. 2. In a second embodiment, the invention relates to the polynucleotide whose sequence is between position 927 and position 2368 of SEQ ID No. 1. This polynucleotide encodes the mature polypeptide of Penicillium funiculosum after cleavage of the signal peptide. The invention also relates to polynucleotides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% and preferably at least 99% identity with the polynucleotide whose sequence is between position 845 and position 2368 of SEQ ID No. 1 and / or with the polynucleotide whose sequence is between position 927 and position 2368 of SEQ ID No. 1. These polynucleotides encode an α-arabinofuranosidase activity B. Preferably , these polynucleotides encode an aL-arabinofuranosidase B of Penicillium funiculosum. The term "identical nucleotides" means nucleotides that do not vary or do not change between two sequences. These polynucleotides can have a deletion, an addition or a substitution of at least one nucleotide compared to the reference polynucleotide. The invention also relates to polynucleotides having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% and preferably at least 99% similarity to the polynucleotide whose sequence is between position 845 and position 2368 of SEQ ID No. 1 and / or with the polynucleotide whose sequence is between position 927 and position 2368 of SEQ ID No. 1. These polynucleotides encode an α-arabinofuranosidase activity B. Preferably , these polynucleotides encode an aL-arabinofuranosidase B of Penicillium funiculosum. Expression similarity means the measurement of the similarity between the nucleic or protein sequences. These polynucleotides can have a deletion, an addition or a substitution of at least one nucleotide compared to the reference polynucleotide. The degree of similarity between two sequences, quantified by a result, is based on the sequence identity in percentage and / or the substitution that the sequence retains. Methods for measuring and identifying the degree of similarity between nucleic acid sequences are well known to those skilled in the art. It is possible to use, for example, Vector NTi Vector NTi 9.1.0, an alignment program AlignX (Clustal W algorithm) (Invitrogen INFORMAX http: // ww. Invitrogen. Com). Preferably, the default parameters are used. Preferably, polynucleotides having a degree of similarity to a reference polynucleotide retain the function of the reference sequence. In the present case, the polynucleotides encode an α-L-arabinofuranosidase B activity. The invention also relates to polynucleotides capable of hybridizing selectively with the polynucleotide whose sequence is between position 845 and position 2368 of SEQ ID No. 1 and / or with the polynucleotide whose sequence is between position 927 and position 2368 of SEQ ID No. 1. Preferably, selective hybridization is carried out under conditions of average severity and preferably under very severe conditions. These polynucleotides encode an α-L-arabinofuranosidase B activity. Preferably, these polynucleotides encode an α-L-arabinofuranosidase B from Penicillium funiculosum. The term "sequence capable of selectively hybridizing" means, according to the invention, the sequence that hybridizes to the reference sequence at a level significantly above the background noise. The level of the signal generated by the interaction between the sequence capable of selectively hybridizing and the reference sequences is generally 10 times, preferably 100 times more intense than that of the interaction of the other DNA sequences generating the background noise. The severe hybridization conditions that allow for selective hybridization are well known to those skilled in the art. In general, the hybridization and the washing temperature is at least 5 ° C lower than the Tm of the reference sequence at a given pH and for a given phonic concentration. Typically, the hybridization temperature is at least 30 ° C for a polynucleotide of 15 to 50 nucleotides and at least 60 ° C for a polynucleotide of more than 50 nucleotides. By way of example, hybridization is carried out in the following buffer: 6 x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA , 500 iag / ml of denatured salmon sperm DNA. The washings are carried out, for example, successively at a low severity level in a 2x SSC buffer, 0.1% SDS, at an average severity level in a 0.5x SSC buffer, 0.1% SDS and at a high severity level in a buffer. O.lx SSC buffer, 0.1% SDS. Hybridization can be carried out, of course, according to other common methods well known to those skilled in the art (see in particular Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989. Preferably, the polynucleotides are selectively hybridize with a reference polynucleotide which retains the function of the reference sequence In the present case, the polynucleotides, which selectively hybridize with the polynucleotide whose sequence is between position 845 and position 2368 of SEQ ID No. 1 and / or with the polynucleotide whose sequence is between position 927 and position 2368 of SEQ ID No. 1, encode an aL-arabinofuranosidase B activity.

The invention relates generally to polynucleotides that encode polypeptides according to the invention. Due to the degeneracy of the genetic code, several polynucleotides can encode the same polypeptide. Another object of the present invention is a polynucleotide whose sequence is represented in SEQ ID No. 1. The polynucleotide of SEQ ID No. 1 comprises sequences that flank the open reading frame (ORF) of the abfB-1 gene of Penicillium funiculosum . They are in particular promoter and terminator sequences of the abfB-1 gene. The abfB gene can be expressed from its homologous regulatory sequences, in particular for overexpression in Penicillium funiculosum or in other filamentous fungi. In another embodiment, the abfB gene can be expressed in various host organisms such as bacteria, yeast and fungi, for example. The abfB gene can be expressed in a host organism under the control of the promoter of SEQ ID No. 1 of the present invention or under the control of a heterologous promoter. Expression cassettes According to one embodiment of the invention, a polynucleotide encoding a polypeptide according to the invention is inserted into an expression cassette using cloning techniques well known to those skilled in the art. This expression cassette comprises the elements necessary for transcription and translation of the sequences encoding the polypeptides according to the invention. Advantageously, this expression cassette comprises both elements that make it possible to cause a host cell to produce a polypeptide and elements necessary for the regulation of this expression. These expression cassettes comprise, in the direction of transcription: a promoter that is functional in a host organism; a polynucleotide according to the invention; a terminator sequence that is functional in the same host organism. Any type of promoter sequence can be used in the expression cassettes according to the invention. The choice of promoter will depend in particular on the host organism chosen for the expression of the gene of interest. Some promoters allow a constitutive expression while other promoters are conversely inducible. Among the promoters that are functional in fungi, mention may be made in particular of the glyceraldehyde-3-phosphate dehydrogenase of Aspergillus nidulans (Roberts et al., Current Genet, 15: 177-180, 1989). Among the promoters that are functional in bacteria, we can mention in particular that for the bacteriophage T7 RNA polymerase (Studier et al., Methods in enzymology 195: 60-89, 1990). Among the promoters that are functional in yeast, the promoter for the GALI gene (Elledge et al., Proc. Nati, Acad. Sciences, USA, 88: 1731-1735, 1991) or the promoters S. cerevisiae GAL4 and ADH. All of these promoters are described in the literature and are well known to those skilled in the art. For expression in Penicillium funiculosum, expression cassettes comprising a histone H4B promoter, an aspartylcoprotease acid promoter or a csll3 promoter (WO 00/68401) will be chosen, for example. The expression cassettes according to the present invention may additionally include any other sequence necessary for the expression of the polypeptides or polynucleotides, such as for example, the regulatory elements or signal sequences that allow the secretion of the polypeptides produced by the organism. Guest. It is possible to use in particular any regulatory sequence that makes it possible to increase the level of expression of the coding sequence inserted in the expression cassette. In accordance with the invention, it is possible to use in particular, a combination with the regulatory promoter sequence, other regulatory sequences, which are located between the promoter and the coding sequence, such as the transcription activators. A wide variety of terminator sequences can be used in the expression cassettes according to the invention, these sequences allow the termination of transcription and polyadenylation of mRNA. Any terminator sequence that is functional in the selected host organism can be used. For expression in Penicillium funiculosum, for example, expression cassettes comprising a histone terminator H4.B, a terminator of the aspartyl acid protease or a csll3 terminator (WO 00/68401) will be chosen.

The object of the present invention is also a polynucleotide comprising an expression cassette according to the invention, advantageously the expression cassettes according to the present invention are inserted into a vector. The present invention therefore also relates to expression or replication vectors for transforming a host organism comprising at least one polynucleotide or an expression cassette according to the present invention. This vector may correspond in particular to a plasmid, a cosmid, a bacteriophage or a virus in which a polynucleotide or an expression cassette according to the invention has been inserted. The techniques for constructing these vectors and for inserting a polynucleotide of the invention into these vectors are well known to those skilled in the art. In general, it is possible to use any vector capable of being maintained, auto-replicated or propagated in a host cell to induce in particular the expression of a polynucleotide or of a polypeptide. Those skilled in the art will choose the appropriate vectors according to the host organism to be transformed, and in accordance with the transformation technique used. The vectors of the present invention are used in particular to transform a host organism for vector replication and / or the expression of a polypeptide according to the invention in the host organism. The invention also relates to a method for preparing a polypeptide according to the invention comprising the following steps: a host organism is transformed with an expression vector comprising an expression cassette according to the invention and / or with a polynucleotide according to the invention, the polypeptides produced by the host organism are isolated. Host Organisms The object of the present invention is also a method for transforming a host organism by integrating into said host organism at least one polynucleotide or an expression cassette or a vector according to the invention. The polynucleotide can be integrated into the genome of the host organism or can be replicated stably in the host organism. Methods for transforming host organisms are well known to those skilled in the art and are described in the literature. The present invention also relates to a host organism transformed with a polynucleotide, an expression cassette or a vector according to the invention. The expression host organism means, in particular, according to the invention, any mono- or multicellular organism, lower or higher, selected from bacteria, yeasts and fungi. The host organism of expression means a non-human organism. Advantageously, the yeasts are chosen from Pichia pastoris, Saccharomyces cerevisae, Yarrowia lipolytica and Schwanniomyces occidentalis. The fungi are chosen from Aspergillus and Penicillium, preferably between Penicillium funiculosum, Trichoderma reesei, Aspergillus niger, Aspergillus awamori, Aspergillus kawachii and Trichoderma koningii. In a preferred embodiment, the host organism is a strain of Penicillium funiculosum wherein an ABFB polypeptide is expressed or overexpressed in accordance with the invention. Techniques for constructing vectors, transforming host organisms and expressing heterologous proteins in these organisms are widely described in the literature (Ausubel, FM et al., "Current Protocols in Molecular Biology", Volumes 1 and 2, Greene Publishing Associates and Wiley Interscience, 1989, T. Maniatis, EF Fritsch, J. Sambrook, "Molecular Cloning: A Laboratory Handbook", 1982). Additives for food and feed for animals The present invention therefore relates to food additives that provide an activity of α-L-arabinofuranosidase B. The intake of this type of enzymatic activity makes it possible to increase the digestibility of the food and increase its nutritional value. The term nutritional additive means a substance that is added intentionally to a food, generally in small quantities, to improve its nutritional characteristics or its digestibility. Nutritional additives for animals may contain, for example, vitamins, mineral salts, amino acids and enzymes.

Typically, nutritional additives for animals comprise a polypeptide according to the invention, a host organism according to the invention or a fermentation broth of a host organism according to the invention. Thus, polypeptides having an α-L-arabinofuranosidase B activity according to the invention can be purified or isolated from a strain of Penicillium funiculosum or from a recombinant host organism for the preparation of a nutritional additive for animals. Alternatively, a strain of Penicillium funiculosum or a host organism producing AbfB polypeptides can be used directly for the preparation of a nutritional additive for animals. In a preferred embodiment of the invention, the culture supernatant or fermentation broth of a strain of Penicillium funiculosum or a host organism according to the invention is used for the preparation of nutritional additives for animals. This modality is particularly advantageous when the ARFE polypeptides are secreted by the Penicillium funiculosum strain or the host organism. Commonly, this culture supernatant is concentrated or lyophilized for the preparation of the nutritional additive. Accordingly, the invention also relates to a method for preparing an ABFB enzyme comprising the following steps: a) culturing a strain of Penicillium funiculosum or a transformed host organism according to the invention under conditions to induce the expression of ABFBs, b ) separate the culture supernatant comprising the ABFB enzyme. This culture supernatant or fermentation broth can then be concentrated or lyophilized for the formulation of an additive for food or an animal feed. If the host organism does not secrete the ABFB enzyme into the culture medium, an additional step of opening the cells and purifying the cell extract may be necessary. The nutritional additives of the present invention comprise an α-L-arabinofuranosidase B activity but may also comprise other nutritional substances such as vitamins, amino acids or mineral salts. The additives according to the invention increase the digestibility of animal feeds, thus contributing to increase the nutritional value of cereal-based diets (wheat, barley, corn, oats, rye and the like) and in particular in oil cakes ( soybean, sunflower, rapeseed and the like). The present invention also relates to animal feeds comprising a nutritional base and a nutritional additive according to the invention. These animal feeds are commonly provided in the form of meals or granules in which the additives according to the invention are incorporated. The term animal feed means anything that serves as animal feed. The animal feeds comprise a polypeptide according to the invention, a host organism according to the invention or a fermentation broth of a host organism according to the invention. For intensive animal husbandry, these animal feeds commonly comprise a nutritional base and nutritional additives. The term "nutritional base" means that it constitutes the main part of the feed ration of animals, which consists, by way of example, of a mixture of cereals, proteins and fat of animal and / or vegetable origin. The nutritional bases for animals are suitable as foods for these animals and are well known to those skilled in the art. Commonly, these nutritional bases comprise, for example, corn, wheat, peas and soybeans. These nutritional bases are adequate for the requirements of the various animal species for which they are intended. These nutritional bases may already contain nutritional additives such as vitamins, mineral salts and amino acids. In a preferred embodiment, the invention relates to animal feed for monogastric animals and in particular for poultry and pigs. Poultry comprise, in particular, laying hens, broiler chickens, turkeys and ducks. The pigs comprise in particular the growing and finishing pigs and the piglets. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Determination of the optimum pH of the enzyme ABFB-1 in a series of Mcllvaine buffer (pH 2.2 to 8) at 40 ° C in the presence of 5 mM PNPAF. Figure 2: Determination of the optimum temperature for the ABFB-1 enzyme at its optimum pH in the presence of 5 mM PNPAF. Figure 3: Determination of kinetic constants Km and Vm (l / vi = f (l / S) for ABFB-1 for a range of PNPAF from 0.5 mM to 5 mM at pH 3.4 and 60 ° C. Figure 4: Values of Relative quantitative differential expression of the abfB-1 and abfB-2 genes according to the culture conditions of P. funiculosum DETAILED DESCRIPTION OF THE INVENTION E emplos Development of the assay of L-arabinofuranosidase B activity The L-activity was measured arabinofuranosidase B from a culture of P. funiculosum in M2 medium with a mixed addition composed of 0.15% provasoy and 0.3% cellulose after 40 hours.The samples were collected at 48 hours and 72 hours of culture. performed in a 200 ml Erlenmeyer flask with a useful volume of 50 ml The activity was determined by hydrolyzing 5 mM para-nitrophenyl- (L-arabinofuranoside (PNPAF) in a 50 mM sodium acetate buffer, pH 5. They were incubated 50 μl of culture supernatant with 250 μl of pre-heated substrate at 50 ° C before 15 min The reaction was stopped by adding 500 μl of 0.5 M NaOH. The release of p-nitrophenyl (PNP) is measured at 405 nm with a molar extinction coefficient of 17,000 M-l. cm-l. One unit of enzyme is defined as the amount of enzyme that hydrolyzes 1 thiol of PNPAF per minute under the conditions described above. For the culture of P. funiculosum, we obtained 20 mU.ml-1 after 48 hours and 112 mU.ml-1 after 72 hours of culture. These results are in agreement with the literature, indeed for Aspergillus niger, activities of the order of 100 to 600 mU.ml-1 were observed according to the inductor used in the culture. Cloning of the abfB ORF of P. funiculosum in Saccharomyces cerevisiae Starting with P. funiculosum genomic DNA, the abfB gene was amplified by PCR with the help of the primer pair (HindIII-abfB / Xbal-abfB) under the following conditions (94 ° C 30 sec; 62 ° C 30 sec; 1 min 30 sec at 72 ° C) for 30 cycles. The PCR product was cloned into a commercial vector pGEM-T (tm) easy. Sequence of the pair of PCR primers: Xbal-abfB-l: > 5 '-TCTAGAATGTTTCCAAGAATAAAACCAG-3' < HindIII-abfB-l: > 5 '-AAGCTTTCATGCAAAGGCAGTCT-3' < The 1534 bp HindIII / Xbal fragment was removed from the pGEM-T vector and subcloned into the HindIII / Xbal sites in a pJL 52 transport vector (placl95-PGK / CYCl). For heterologous expression, the abfB gene is therefore under the control of the constitutive PGK promoter of the gene encoding phosphoglycerate kinase (S. cerevisiae) and the CYC1 terminator (S. cerevisiae) of the gene encoding a cytochrome C activity oxidase The new expression cassette is called pOT-01. The strain s. cerevisiae JF # 1194 (CEN PK113-5D), a clone derived from the strain CEN.PK 122 carrying the auxotrophy ura 3-52, was transformed (lithium acetate / thermal shock method) with the expression vector pOT- 01. The transformant strains were selected by complementation of the phenotype in uracil free selective plates (URA3 marker). The six transformants were selected to prove the presence of an arabinofuranosidase B activity in the culture supernatant. The transformants were cultured in 50 ml of uracil-free YNB medium (except the wild-type control strain) for 24 hours. The arabinofuranosidase B activity was assayed in the culture supernatants with the aid of the method described in the preceding paragraph. Determination of the optimum pH The abfB-1 gene that codes for an arabinofuranosidase B activity derived from P. funiculosum was cloned in S. cerevisiae. After controlling for the presence of an arabinofuranosidase B activity in several transformants, a transformant was chosen and the ABFB activity was tested in the culture supernatant after 24 hours of culture. The cultures were carried out in a 200 ml Erlenmeyer flask (working volume 50 ml). The activity was determined in the presence of 4 mM of p-nitrophenyl-a-L-arabinofuranoside (PNPAF) in a series of Mcllvaine buffer (pH 2.2 to 8.0). 80 μl of culture supernatant was incubated with 320 μl of preheated substrate at 40 ° C for 10 min. The reaction was stopped by adding 1 ml of Na 2 CO 3 IM. The release of p-nitrophenyl is measured at 405 nm. "1 An enzyme unit is defined as the amount of enzyme that hydrolyzes 1 μmol of PNPAF per minute under the conditions defined above.The activity curve is depicted in Figure 1 For ABFB-1, the activity optimum is at pH 3.4 and the enzyme retains 65% activity at pH 5. Determination of the optimal temperature Using the same protocol, we determine the optimal temperature for ABFB-1 activity. The enzyme was incubated for 10 min at each of the temperatures in a Mcllvaine buffer at pH 3.4 The activity curve is presented in Figure 2. The ABFB-1 of P. funiculosum has an activity optimum at 60 ° C. The ABFB-1 therefore has a higher temperature optimum than the ABFBs described, if the optimal temperature and pH of the enzyme ABFB-1 (pH 3.4 and 60 ° C) are selected, it is observed that the activity for ABFB-1 1 is 4 times as high as the activity determined in a buffer acetat or at pH 5 and 40 ° C (424 mU vs. 102 mU). Determination of Km and Vm The kinetic constants (Km and Vm) for ABFB-1 were determined by measuring the hydrolysis of PNPAF over time, under the optimal conditions determined above. The substrate concentration ranges (PNPAF) were established between 0.5 and 5 mM in a buffer at pH 3.4. The hydrolysis kinetics were monitored for 10 minutes at 60 ° C. The results were treated according to the double inverse method (Lineweark and Burk) and are presented in Figure 3. The Km value is lmM for ABFB-1. In comparison, in the literature, Km values for this type of enzyme vary from 0.05 to 1.2 mM according to the genus and the fungal species studied. The ABFB-1 has a maximum hydrolysis rate (Vm) of 521 moles of PNPAF / mole of enzyme / min under the conditions described above. Determination of the molecular weight of the ABFB-1 enzyme To determine the molecular weight of the enzyme ABFB-1, the culture supernatant, derived from the culture of a mutant (S. cerevisiae) in a minimal medium, was concentrated 200 times, denatured by boiling at 100 ° C for 5 min and then deposited on an SDS-polyacrylamide gel. It is observed that the amount of extracellular proteins is extremely low in the wild-type strain. For mutants, the ABFB-1 enzyme is secreted into the culture supernatant. It is predominant in relation to the basal level of extracellular proteins of S. cerevisiae. The determination of the molecular weight was carried out with the help of the SeeBlue size marker (Invitrogen). The results are presented in Table 1.

Table 1: Molecular weight of ABFB-1 in KDa. We compared the molecular weight predicted by the Vector NTi algorithm and the weight obtained by electrophoretic migration on a denaturing SDS-PAGE gel. We observed an overestimation of the molecular weight of the enzyme in the SDS-PAGE gel. A high glycosylation of the enzyme is actually suggested by the visualization in gel of a diffuse electrophoretic band (glycosylations 0 and N). Glycosylation occurs during the processing of proteins in the expressing organism. Analysis of the expression profile of the abfB-1 gene in Penicillium funiculosum Penicillium funiculosum has two genes that code to the α-L-arabinofuranosidases B: the abfB-1 and abfB-2 genes. The expression profiles of these genes were compared under various culture conditions of P. funiculosum. P. funiculosum was cultured under conditions to induce cellulolytic and hemicellulolytic enzymes (industrial culture medium type M2) and under non-producing conditions (minimum glucose medium MO). After 40 hours of culture, the cultures were stopped, the mycelium was recovered and the total RNAs were extracted. The quantity and quality of the RNAs were evaluated by measuring the absorbance at 260 nm and at 280 nm (ratio 260/280 >; 1.8). The level of transcripts encoding arabinofuranosidase type B activities (ABFB-1 and ABFB-2) was quantified under each of the two conditions (MO and M2) by quantitative real-time PCR. The gene encoding tubulin from P. funiculosum (tub-1) was used as a control under both conditions. This gene encodes a structural protein that is essential for the integrity of the cell. This gene is commonly used as the reference gene because it presents a constant level of expression regardless of the culture condition used (ubiquitous). The specific primers for quantitative PCR were designated for each of the genes (abfB-1, abfB-2 and tub-1). For the two growth conditions (MO and M2), 2 μg of total RNA was retrotranscribed. A series of dilutions of the complementary DNAs derived from the retrotranscription were carried out to determine the optimal conditions for the amplification of the target genes (restrictions of the quantitative PCR method and for the efficiency of these primer pairs). The normalized results are presented in Table 2 and Figure 4.

Table 2: Values for the differential expression of the abfB-1 and abfB-2 genes as a function of the culture conditions of P. funiculosum. The transcriptional regulations of the genes coding for cellulolytic and hemicellulolytic activities have been described. The expression of these genes depends very much on the nature and / or complexity of the carbon and nitrogen source in which the microorganism is grown.

A high transcriptional repression of these genes in the presence of glucose has been reported. This regulation is carried out through a CreA catabolic repression protein that binds specifically to the promoter of these genes and blocks their transcription. In our experiment to quantify, by PCR, the abfB-1 and abfB-2 messengers, it was determined that the level of expression of these two genes under the glucose (MO) condition is very low. This is consistent with the literature, since it has been shown that these genes have a basal level of expression even under unfavorable conditions (absence of cellulolytic and / or hemicellulolytic substrates). The results obtained for the MO condition agree with the literature. With regard to the expression of the abfB-1 gene, an induction factor is observed that is 107 times as high as the basal level obtained under the MO condition, while the abfB-2 gene is not overexpressed. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A polypeptide adapted for use in animal feed, characterized in that it comprises a polypeptide chosen from the following polypeptides: the polypeptide of SEQ ID No. 2 , the polypeptide whose sequence is between position 28 and position 507 of SEQ ID No. 2, a fragment of the polypeptide of SEQ ID No. 2 having an α-arabinofuranosidase activity B, 2. Polypeptide characterized in that it has a aL-arabinofuranosidase B activity and having an identity of at least 90% with the polypeptide of SEQ ID No.
2.
3. Polynucleotide that encodes an aL-arabinofuranosidase B activity, characterized in that it is chosen from the following polynucleotides: polynucleotide whose sequence is between position 845 and position 2368 of SEQ ID No. 1, the polynucleotide whose sequence a is between position 927 and position 2368 of SEQ ID No. 1, a polynucleotide encoding a polypeptide according to claim 1.
4. Polynucleotide, characterized in that it has the sequence of SEQ ID No. 1 or the sequence complementary to SEQ ID No. 1.
5. Cassette of expression, characterized in that it comprises, in the direction of transcription: a promoter that is functional in a host organism; a polynucleotide according to claim 2; and - a terminator sequence that is functional in the same host organism.
6. Vector characterized in that it comprises a polynucleotide according to one of claims 2 to 3 and / or an expression cassette according to claim 4.
7. A host organism characterized in that it is transformed with a polynucleotide according to one of the claims 2 to 3, an expression cassette according to claim 4 and / or a vector according to claim 5.
8. Host organism according to claim 6, characterized in that it is chosen from yeasts and filamentous fungi.
9. Host organism according to claim 7, characterized in that it is a strain of Penicillium funiculosum.
10. Nutritional additive for animals, characterized in that it comprises a polypeptide according to claim 1.
11. Nutritional additive for animals, characterized in that it comprises a host organism according to one of claims 6 to 8 and / or a fermentation broth of A host organism according to one of claims 6 to 8.
12. The nutritional additive for animals according to claim 9, characterized in that it is in liquid form or in powder form.
13. Animal feed, characterized in that it comprises a nutritional base for animals and a nutritional additive for animals according to one of claims 9 to 11.
14. Use of a polypeptide according to claim 1 or of a host organism in accordance with with one of claims 6 to 8 for the preparation of a nutritional additive for animals or an animal feed.
15. Use of a polypeptide according to claim 1 or of a host organism according to one of claims 6 to 8, for hydrolyzing the α-L-arabinofuranosyl bonds of the arabinofuranosyl-oligosaccharide compounds.